Figure 1.

Interaction of human p57 with PCNA. (A) Yeast HF7c cells were simultaneously transformed with a plasmid expressing a GAL4^bd fusion protein and a plasmid expressing a GAL4^ad fusion protein as indicated. Cells were streaked on nonselective medium with histidine (-Leu, -Trp), selective medium without histidine (-Leu, -Trp, -His), and selective medium without histidine but containing 5 mM 3-amino-1,2, 3-triazole (-Leu, -Trp, -His, 3-AT). Staining for β-galactosidase expression, activated from an independent GAL4 responsive promoter, is shown (β-gal, Lower Right). The C-terminal domain of p21 possesses as a trans-activating activity (self-activation) when expressed as a fusion protein with the GAL4 DNA binding domain. (B) Equal amounts of histidine-tagged human p57C (lane 1), mouse p57 (lane 2), human p27 (lane 3), full-length human p57 (lane 4), or human p21 (lane 5) were incubated with 2 μg purified human PCNA protein. Mixtures were recovered on Ni-Sepharose beads, resolved by SDS/PAGE, and stained with Coomassie blue. (C) Exponentially growing Sf9 cells were singly infected with baculoviruses expressing the histidine-tagged C-terminal domain of human p57 (His-p57C), full-length human p57 (hp57), human PCNA, or doubly infected with their combinations as indicated on the top of each lane. Cells were metabolically labeled with [³⁵S]methionine 40 h postinfection and p57 protein complexes were recovered on Ni-Sepharose or immunoprecipitated with an anti-p57 antibody and analyzed by SDS/PAGE and autoradiography. (D) Exponentially growing Sf9 cells were coinfected with baculoviruses expressing various human proteins. CDK4 and cyclin D1 protein complexes were immunoprecipitated with indicated antibodies and analyzed by SDS/PAGE and autoradiography.